Reduced dipeptide analogues as calcium channel antagonitsts

ABSTRACT

The present invention provides novel N-type calcium channel blockers, compositions comprising them, and methods of their use. The compounds of the invention are useful in the treatment of stroke, cerebral ischemia, pain, epilepsy, and head trauma. The novel compounds provided by this invention have the Formula I:  
                 
 
     wherein  
     R 1  is H or methyl,  
     R 2  is H, azepanylcarbonyl, C 1 -C 7  alkyl, —(CH 2 ) n -phenyl, wherein the phenyl is unsubstituted or substituted with C 1 -C 4  alkyl, C 1 -C 4  alkoxy, or halo,  
     R 3  is C 1 -C 5  alkyl,  
     X is —NR 4 R 5  or —OR 7 ,  
     R 4  and R 5  are independently H, C 1 -C 5  alkyl, or  
     R 4  and R 5  together with the nitrogen to which they are both bound form:  
                 
 
      R 6  is —(CH 2 ) n -phenyl, wherein the phenyl is unsubstituted or substituted with C 1 -C 4  alkyl, C 1 -C 4  alkoxy, or halo,  
     A and B are independently —CO— or —CH 2 —, provided that A and B are not both —CO—,  
     R 7  is C 1 -C 5  alkyl,  
     Z is —CH 2 —, —O—, —S—, or —N(R 8 )—,  
     R 8  is H or C 1 -C 6  alkyl, and  
     n is  1  or  2.

FIELD OF THE INVENTION

[0001] The present invention relates to novel compounds, pharmaceuticalcompositions thereof and methods of using them in the treatment ofstroke, cerebral ischemia, pain, epilepsy, and head trauma. Inparticular, the present invention relates to reduced backbone dipeptidecompounds that are potent antagonists of N-type calcium channels.

SUMMARY OF THE RELATED ART

[0002] The entry of excessive amounts of calcium ions into neuronsfollowing an ischemic episode or other neuronal trauma has been welldocumented. Uncontrolled high concentrations of calcium in neuronsinitiate a cascade of biochemical events that disrupts normal cellularprocesses. Among these events are the activation of proteases andlipases, breakdown of neuronal membranes, and the formation of freeradicals, which may ultimately lead to cell death. Several types ofcalcium channels have been discovered: the L, N, P, Q, R, and T types.Each type possesses distinct structural features, functional properties,and cellular/subcellular distributions. N-type calcium channels aretissue specific, restricted to the central and peripheral neurons of theforebrain and areas rich in synaptic connections. They have well definedroles, e.g., regulation of calcium flux necessary fordepolarization-evoked release of transmitter from synaptic endings; andthey can be selectively blocked by high-affinity ligands, likeω-conotoxins and synthetic analogs. Bowersox S. S., et al., Drug Newsand Perspective, 1994;7:261-268.

SUMMARY OF THE INVENTION

[0003] The present invention provides novel compounds and compositionsthat are capable of blocking N-type calcium channels. The compounds areuseful in a method for treating afflictions associated with increasedcalcium ion uptake through N-type calcium channels, including suchsystemic effects as stroke, cerebral ischemia resulting from cardiacarrest, head trauma, closed head injury, pain, and epilepsy. Furtheradvantages of this invention will be clear to one skilled in the artfrom the reading of the description that follows.

[0004] The present invention comprises new compounds and, moreparticularly, novel reduced backbone dipeptide compounds that are usefulas N-type calcium channel blockers in mammals. The novel compounds ofthe present invention are represented by the following structuralFormula I:

[0005] wherein A, B, X, R¹, R², R³, and R⁶ are defined in more detailbelow and generally form a reduced dipeptide backbone.

[0006] The invention also includes novel compositions of mattercontaining the above-defined compound that are useful as neuroprotectiveagents for the treatment of afflictions associated with increasedcalcium ion uptake through N-type calcium channels in mammals, as wellas the methods of treatment using such compositions.

[0007] The foregoing merely summarizes certain aspects of the presentinvention and is not intended, nor should it be construed, as limitingthe invention in any manner. All patents and other publicationsreferenced herein are hereby incorporated by reference in theirentirety.

DETAILED DESCRIPTION OF THE INVENTION

[0008] The present invention comprises novel reduced backbone dipeptidecompounds and pharmaceutical compositions thereof that are useful asN-type calcium channel antagonists.

[0009] The novel compounds of the present invention have the followinggeneric structural Formula I:

[0010] wherein

[0011] R¹ is H or methyl,

[0012] R² is H, azepanylcarbonyl, C₁-C₇ alkyl, —(CH₂)_(n)-phenyl,wherein the phenyl is unsubstituted or substituted with C₁-C₄ alkyl,C₁-C₄ alkoxy, or halo,

[0013] R³ is C₁-C₅ alkyl,

[0014] X is —NR⁴R⁵ or —OR⁷,

[0015] R⁴ and R⁵ are independently H, C₁-C₅ alkyl, or

[0016] R⁴ and R⁵ together with the nitrogen to which they are both boundform:

[0017]  R⁶ is —(CH₂)_(n)-phenyl, wherein the phenyl is unsubstituted orsubstituted with C₁-C₄ alkyl, C₁-C₄ alkoxy, or halo,

[0018] A and B are independently —CO— or —CH₂—, provided that A and Bare not both —CO—,

[0019] R⁷ is C₁-C₅ alkyl,

[0020] Z is —CH₂—, —O—, —S—, or —N(R⁸)—,

[0021] R⁸ is H or C₁-C₆ alkyl, and

[0022] n is 1 or 2.

[0023] In a preferred embodiment of the compound of Formula I,

[0024] R⁴ and R⁵ are independently H, C₁-C₅-alkyl, or

[0025] R⁴ and R⁵ taken together with the nitrogen to which they are bothbound form 1-pyrrolidinyl, 1-piperidinyl, or 1-azepanyl, or

[0026] R⁴ and R⁵ taken together with the nitrogen to which they are bothbound are:

[0027] and

[0028] Z is —S—or —O—.

[0029] Also provided are pharmaceutically acceptable salts, esters,amides, and pro-drugs of the compounds of the Formula I.

[0030] The term “alkyl” means a straight or branched chain hydrocarbon.Representative examples of alkyl groups are methyl, ethyl, propyl,isopropyl, isobutyl, butyl, tert-butyl, sec-butyl, pentyl, hexyl,(CH₃)₂CHCH₂CH₂—, (CH₃)₃CCH₂CH₂—, and heptyl.

[0031] The term “alkoxy” means an alkyl group attached to an oxygenatom. Representative examples of alkoxy groups include methoxy, ethoxy,tert-butoxy, propoxy, and isobutoxy.

[0032] In a preferred embodiment, compounds of the invention areselected from the group consisting of:

[0033]2-((2-(Azaperhydroepinylcarbonylamino)-4-methylpentyl)amino)-N-(tert-butyl)-3-(4-(phenylmethoxy)phenyl)-(2S)-propanamide;

[0034] [S-(R*,R*)]-Azepane-1-carboxylic acid{1-[2-(4-benzyloxy-phenyl)-1-tert-butoxymethyl-ethylcarbamoyl]-3-methyl-butyl}-amide;

[0035] [S-(R*,R*)]Azepane-1-carboxylic acid(1-{[2-(4-benzyloxy-phenyl)-1-tert-butylcarbamoyl-ethylamino]-methyl}-3-methyl-butyl)-amide;

[0036] [S-(R*,R*)]-2-Dimethylamino-4-methyl-pentanoic acid[2-(4-benzyloxy-phenyl)-1-morpholin-4-ylmethyl-ethyl]-amide;

[0037] [S-(R*,R*)]-Azepane-1-carboxylic acid{1-[2-(4-benzyloxy-phenyl)-1-morpholin-4-ylmethyl-ethylcarbamoyl]-3-methyl-butyl}-amide;

[0038] [S-(R*,R*)]Azepane-1-carboxylic acid{1-[2-(4-benzyloxy-phenyl)-1-(tert-butylamino-methyl)-ethylcarbamoyl]-3-methyl-butyl}-amide;

[0039] (S,S)-Azepane-1-carboxylic acid{1-[2-(4-benzyloxy-phenyl)-1-diethylaminomethyl-ethylcarbamoyl]-3-methyl-butyl}-amide;

[0040](S)-2-(2-Amino-4-methyl-pentylamino)-3-(4-benzyloxy-phenyl)-N-tert-butyl-propionamidedihydrochloride; and

[0041]2-(2-Amino-4-methyl-pentylamino)-3-(4-benzyloxy-phenyl)-N-tert-butyl-propionamidedihydrochloride.

[0042] The compounds of the invention may be readily prepared as setforth in the following reaction scheme(s) which employ general syntheticmethods well-known to those skilled in organic chemistry. The followingdefinitions apply: H₂SO₄ Sulfuric acid HBTUO-benzotriazol-1-yl-N,N,N′,N′-tetramethyluronium- hexafluorophosphateDMAP 4-(Dimethylamino)pyridine DMF N,N-Dimethylformamide Et₂ODiethylether HCl Hydrochloric acid NaBH₃CN Sodium cyanoborohydride MeOHMethanol TFA Trifluoroacetic acid DMC 4,4′-Dichloro-α-methylbenzhydroli-Pr₂Net Diisopropyl ethylamine LAH Lithium aluminum hydride HNMeOMeMethoxymethylamine Pd/C Palladium on carbon catalyst

[0043]

[0044] Wherein R^(a)-R^(g) are any chemical moiety consistent with thedefinitions of R¹-R⁷.

[0045] The compounds of the invention are also useful research tools forstudying the biological, cellular effects of blocking N-type calciumchannels.

[0046] The invention further comprises a pharmaceutical composition forthe treatment of illnesses induced by uncontrolled high concentrationsof calcium in neurons, which composition comprises a compound of FormulaI as defined above or, a pharmaceutically acceptable salt, solvent orpro-drug thereof, in an amount effective in the treatment of saidillnesses, and a pharmaceutically acceptable carrier. The invention alsocomprises a pharmaceutical composition for the treatment of afflictionsassociated with increased calcium ion uptake through N-type calciumchannels, including such systemic effects as stroke, pain, cerebralischemia, head trauma, and epilepsy, which comprises a compound ofFormula I as defined above in an amount effective in the treatment ofsaid disorders, and a pharmaceutically acceptable carrier. Preferredcompositions of the invention are those containing preferred compoundsof Formula I as described above. The compounds will be administered tomammals at the rate of about 0.01 to about 200 mg/kg. Typical dailydoses will be about 5 to about 500 mg per patient.

[0047] The term “pro-drug” refers to compounds that are rapidlytransformed in vivo to yield the parent compound of the above formulae,for example, by hydrolysis in blood. A thorough discussion is providedin T. Higuchi and V. Stella, “Pro-drugs as Novel Delivery Systems,” Vol14 of the A.C.S. Symposium Series, and in Bioreversible Carriers in DrugDesign, ed. Edward B. Roche, American Pharmaceutical Association andPergamon Press, 1987, both of which are incorporated herein byreference.

[0048] The invention further relates to a method for the treatment ofillnesses induced by uncontrolled high concentrations of calcium inneurons by administering to a subject in need of such treatment acompound of Formula I as defined above in an amount effective in suchtreatment, and a method for the treatment of stroke, pain, cerebralischemia, head trauma, and epilepsy utilizing a compound of Formula I asdefined above in an amount effective in such treatment. Preferredmethods of the invention are those administering a preferred compound ofthe Formula I as described above.

[0049] The compounds of the present invention may be administered to apatient either alone or as part of a pharmaceutical composition. Thecompositions may be administered to patients either orally, rectally,parenterally (intravenously, intramuscularly, or subcutaneously),intracistemally, intravaginally, intraperitoneally, intravesically,locally (powders, ointments, or drops), or as a buccal or nasal spray.

[0050] Compositions suitable for parenteral injection may comprisephysiologically acceptable sterile aqueous or non-aqueous solutions,dispersions, suspensions or emulsions, and sterile powders forreconstitution into sterile injectable solutions or dispersions.Examples of suitable aqueous and non-aqueous carriers, diluents,solvents or vehicles include water, ethanol, polyols (propyleneglycol,polyethyleneglycol, glycerol, and the like), suitable mixtures thereof,vegetable oils (such as olive oil), and injectable organic esters suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersions, and by the use of surfactants.

[0051] These compositions may also contain adjuvants such as preserving,wetting, emulsifying, and dispensing agents. Prevention of the action ofmicroorganisms can be ensured by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, sorbic acid, andthe like. It may also be desirable to include isotonic agents, forexample sugars, sodium chloride, and the like. Prolonged absorption ofthe injectable pharmaceutical form can be brought about by the use ofagents delaying absorption, for example, aluminum monostearate andgelatin.

[0052] Solid dosage forms for oral administration include capsules,tablets, pills, powders, and granules. In such solid dosage forms, theactive compound is admixed with at least one inert customary excipient(or carrier) such as sodium citrate or dicalcium phosphate or (a)fillers or extenders, as for example, starches, lactose, sucrose,glucose, mannitol, and silicic acid, (b) binders, as for example,carboxymethylcellulose, alignates, gelatin, polyvinylpyrrolidone,sucrose, and acacia, (c) humectants, as for example, glycerol, (d)disintegrating agents, as for example, agar-agar, calcium carbonate,potato or tapioca starch, alginic acid, certain complex silicates, andsodium carbonate, (e) solution retarders, as for example, paraffin, (f)absorption accelerators, as for example, quaternary ammonium compounds,(g) wetting agents, as for example, cetyl alcohol, and glycerolmonostearate, (h) adsorbents, as for example, kaolin bentonite, and (i)lubricants, as for example, talc, calcium stearate, magnesium stearate,solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof.In case of capsules, tablets, and pills, the dosage forms may alsocomprise buffering agents.

[0053] Solid compositions of a similar type may also be employed asfillers in soft and hard-filled gelatin capsules using such excipientsas lactose or milk sugar as well as high molecular weightpolyethyleneglycols, and the like.

[0054] Solid dosage forms such as tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells, such as entericcoatings and others well known in the art. They may contain opacifyingagents, and can also be of such composition that they release the activecompound or compounds in a certain part of the intestinal tract in adelayed manner. Examples of embedding compositions that can be used arepolymeric substances and waxes. The active compounds can also be inmicro-encapsulated form, if appropriate, with one or more of theabove-mentioned excipients.

[0055] Liquid dosage forms for oral administration includepharmaceutically acceptable emulsions, solutions, suspensions, syrups,and elixirs. In addition to the active compounds, the liquid dosageforms may contain inert diluents commonly used in the art, such as wateror other solvents, solubilizing agents and emulsifiers, as for example,ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propyleneglycol, 1,3-butyleneglycol,dimethylformamide, oils (in particular, cottonseed oil, groundnut oil,corn germ oil, olive oil, castor oil, and sesame oil), glycerol,tetrahydrofurfuryl alcohol, polyethyleneglycols and fatty acid esters ofsorbitan or mixtures of these substances, and the like.

[0056] Besides such inert diluents, the composition can also includeadjuvants such as wetting agents, emulsifying and suspending agents,sweetening agents, flavoring agents, and perfuming agents.

[0057] Suspensions, in addition to the active compounds, may containsuspending agents, as for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth,or mixtures of these substances, and the like.

[0058] Compositions for rectal or intravaginal administrations arepreferably suppositories which can be prepared by mixing the compoundsof the present invention with suitable non-irritating excipients orcarriers such as cocoa butter, polyethyleneglycol, or a suppository wax,which are solid at ordinary temperatures but liquid at body temperatureand therefore, melt in the rectum or vaginal cavity and release theactive compound.

[0059] Dosage forms for topical administration of a compound of thisinvention include ointments, powders, sprays, and inhalants. The activecomponent is admixed under sterile conditions with physiologicallyacceptable carrier and any preservatives, buffers, or propellants as maybe required. Ophthalmic formulations, eye ointments, powders, andsolutions are also contemplated as being within the scope of thisinvention.

[0060] The term “pharmaceutically acceptable salts, esters, amides, andpro-drugs” as used herein refers to those carboxylate salts, amino acidaddition salts, esters, amides, and pro-drugs of the compounds of thepresent invention which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of patients without unduetoxicity, irritation, allergic response, and the like, commensurate witha reasonable benefit/risk ratio, and effective for their intended use,as well as the zwitterionic forms, where possible, of the compounds ofthe invention. The term “salts” refers to the relatively non-toxic,inorganic and organic acid addition salts of compounds of the presentinvention. These salts can be prepared in situ during the finalisolation and purification of the compounds or by separately reactingthe purified compound in its free base form with a suitable organic orinorganic acid and isolating the salt thus formed. Representative saltsinclude the hydrobromide, hydrochloride, sulfate, bisulfate, nitrate,acetate, oxalate, valerate, oleate, palmitate, stearate, laurate,borate, benzoate, lactate, phosphate, tosylate, citrate, maleate,fumarate, succinate, tartrate, naphthylate mesylate, glucoheptonate,lactiobionate and laurylsulphonate salts, and the like. These mayinclude cations based on the alkali and alkaline earth metals, such assodium, lithium, potassium, calcium, magnesium, and the like, as well asnontoxic ammonium, quaternary ammonium, and amine cations, including,but not limited to, ammonium, tetramethylammonium, tetraethylammonium,methylamine, dimethylamine, trimethylamine, ethylamine, triethylamine,and the like. See, for example, Berge S. M., et al., “PharmaceuticalSalts,” J. Pharm. Sci., 1977;66:1-19, which is incorporated herein byreference.

[0061] Those skilled in the art are easily able to identify patientshaving a stroke or at risk of having a stroke, cerebral ischemia, headtrauma, or epilepsy. For example, patients who are at risk of having astroke include, but are not limited to, patients having hypertension orundergoing major surgery.

[0062] The invention will be described in greater detail in conjunctionwith the following specific examples. These examples are provided forillustrative purposes only and are not intended, nor should they beconstrued, as limiting the invention in any manner. Those skilled in theart will appreciate that modifications and variations of the followingexamples can be made without exceeding the spirit or scope of thepresent invention and claims.

EXAMPLE 1 [S-(R*,R*)]-Azepane-1-carboxylic acid{1-[2-(4-benzyloxy-phenyl)-1-tert-butoxymethyl-ethylcarbamoyl]-3-methyl-butyl}-amide

[0063]

[0064] Step A

(S)-2-[(Azepane-1-carbonyl)-amino]-4-methyl-pentanoic acid benzyl ester

[0065]

[0066] A solution of triphosgene (15.7 g, 52.9 mmol) in CH₂Cl₂ (600 mL)was cooled to −10° C. under a nitrogen atmosphere. The solution wastreated dropwise with a solution of (S)-2-amino-4-methyl-pentanoic acidbenzyl ester (28.1 g, 0.127 mol) and pyridine (26 mL, 0.32 mol) in 150mL of CH₂Cl₂. The resulting solution was stirred at −10° C. for 90minutes and then treated with a solution of hexamethyleneimine (22 mL,0.38 mmol) in 75 mL of CH₂Cl₂. The resulting solution was stirred for 48hours at room temperature. The reaction mixture was concentrated, andthe residue was dissolved in ether and washed with 1N HCl solution,water, and saturated aqueous CuSO₄ solution. The organic layer was dried(Mg₂SO₄), treated with activated charcoal, and filtered. The filtratewas concentrated to approximately ½ volume and treated with hexane. Theresulting suspension was stored overnight at −10° C. The solid wascollected by filtration, washed with hexane, and dried under vacuum togive (S)-2-[(azepane-1-carbonyl)-amino]-4-methyl-pentanoic acid benzylester as a white solid (38.6 g, 88%); mp 87-88° C.

[0067] APCI-MS m/z 347 (MH⁺).

[0068] Step B

(S)-2-[(Azepane-1-carbonyl)-amino]-4-methyl-pentanoic acid

[0069]

[0070] A solution of(S)-2-[(azepane-1-carbonyl)-amino]-4-methyl-pentanoic acid benzyl ester(38.5 g, 111 mmol) in 600 mL of THF was hydrogenated at 50 psi over 20%Pd/C (2.00 g) for 17 minutes. The reaction mixture was filtered throughcelite and concentrated to dryness. The residue was heated in 50 mL ofhexane. The resulting suspension was cooled, and the solid was collectedby filtration and washed with hexane. The solid was dried at roomtemperature under vacuum to give(S)-2-[(azepane-1-carbonyl)-amino]-4-methyl-pentanoic acid as a whitesolid (26.6 g, 93%); mp 88-89° C. Microanalysis for C₁₃H₂₄N₂O₃:

[0071] Calculated: C, 60.91; H, 9.44; N, 10.93.

[0072] Found: C, 60.99; H, 9.46; N, 10.85.

[0073] Step C

(S)-1-(4-Benzyloxy-benzyl)-2-tert-butoxy-ethylamine

[0074]

[0075] A mixture of 2.573 g (10 mmol)(S)-β-amino-4-(phenylmethoxy)-benzenepropanol (Advanced Chemtech,Louisville, Ky. 40218), 50 mL 2-methylpropene, 2 mL concentratedsulfuric acid, and 50 mL 1,4-dioxane were shaken in a glass pressurevessel at 25° C. for 62 hours. The milky-white suspension was pouredinto a rapidly stirred mixture containing 3.6 g 85% KOH, 75 mLice-water, and 50 mL diethyl ether. After layers separated, the aqueousphase was extracted with two 50 mL portions of ethyl acetate. Thecombined extracts were washed with 100 mL brine, dried over anhydroussodium sulfate, and concentrated in vacuo giving 288 mg of a pale amberoil which was purified by column chromatography (SiO₂) with 12:1chloroform:methanol to give 131 mg (4%) of(S)-1-(4-benzyloxy-benzyl)-2-tert-butoxy-ethylamine as a clear,colorless oil, that was used directly in the next reaction.

[0076] Step D

[0077] A 3° C. solution of 106 mg (0.41 mmol)(S)-2-[(azepane-1-carbonyl)-amino]-4-methyl-pentanoic acid (Example 1,Step B) and 0.137 mL (0.41 mmol) 4-methylmorpholine in 2 mLN,N-dimethylformamide was treated with 165 mg (0.44 mmol)O-benzotriazol-1-yl-N,N,N′,N′-bis(tetramethylene)uroniumhexafluorophosphate (Novabiochem, La Jolla, Calif.), and stirred for 30minutes, at which time a solution of(S)-1-(4-benzyloxy-benzyl)-2-tert-butoxy-ethylamine in 3 mLdimethylformamide was added and the mixture stirred at 3° C. for 40minutes. The clear solution was poured into 15 mL diethyl ether andwashed successively with 15 mL of each of the following: 5% aqueous HCl,brine, saturated aqueous NaHCO₃, brine (twice) then dried over anhydrousmagnesium sulfate. The solvent was removed in vacuo affording 180 mg ofwhite solid, which was recrystallized from hexane to give 1 16 mg (51%)of the title compound as a white solid; mp 98-99° C. APCI-MS: m/z 552.0[MH⁺].

EXAMPLE 2 [S-(R*,R*)]Azepane-1-carboxylic acid(1-{[2-(4-benzyloxy-phenyl)-1-tert-butylcarbamoyl-ethylamino]-methyl}-3-methyl-butyl)-amidedihydrochloride salt

[0078]

[0079] Step A

N-tert-Butoxycarbonyl-L-leucinal

[0080]

[0081] N-tert-Butoxycarbonyl-L-leucinal was synthesized according to theprocedure published in Goel, et al., Org. Syn., 1988;67:69.

[0082] Step B

(S)-[2-[(1,1-Dimethylethyl)amino]-2-oxo-1-(phenylmethyl)ethyl]-carbamicacid 1,1-dimethylethyl ester

[0083]

[0084] A solution of N-(tert-butyloxycarbonyl)-O-benzyl-L-tyrosine(Bachem, 2.00 g, 5.38 mmol) in 20 mL of DMF was cooled to 0° C. andtreated with iPr NEt (1.5 mL) followed byO-benzotriazol-1a-yl-N,N,N′,N′-bis(tetramethylene)-uronium hexafluorophosphate (2.04 g, 5.38 mmol, Novabiochem, La Jolla, Calif.). Theresulting suspension was stirred for 30 minutes at 0° C. and thentreated with tert-butylamine (0.48 g, 6.56 mmol). The reaction mixturewas stirred for 1 hour at 0° C. and warmed to room temperature. Thereaction mixture was poured into Et O and washed sequentially withsaturated aqueous NaHCO₃ solution, and saturated aqueous NaCl solution.The organic phase was dried (MgSO₄), filtered, and concentrated. Thecrude residue was purified by chromatography (silica gel, 3:1heptane/ethyl acetate) to give(S)-[2-[(1,1-dimethylethyl)amino]-2-oxo-1-(phenylmethyl)ethyl]-carbamicacid 1,1-dimethylethyl ester (2.65 g). MS (CI) 427 (MH).

[0085] Step C

(S)-2-Amino-3-(4-benzyloxy-phenyl)-N-tert-butyl-propionamide

[0086]

[0087] A solution of(S)-[2-(4-benzyloxy-phenyl)-1-tert-butylcarbamoyl-ethyl]-carbamic acidtert-butyl ester (6.0 g, 14.1 mmol, Example 2, Step B) in CH₂Cl₂ (28 mL)was treated with trifluoroacetic acid (28 mL). The resulting solutionwas stirred for 20 minutes and then concentrated. The residue wasdiluted with EtOAc (300 mL), washed with saturated bicarbonate solution(2×300 mL) and brine (300 mL), dried over Na₂SO₄, and concentrated togive 4.2 g (91%) of(S)-2-amino-3-(4-benzyloxy-phenyl)-N-tert-butyl-propionamide. MS: 328(M⁺¹ for C₂₀H₂₆N₂O₃). TLC: Silica gel, R 0.43 (10% MeOH/CHCl).

[0088] Step D

[S-(R*,S*)]-(1-{[2-(4-Benzyloxy-phenyl)-1-tert-butylcarbamoyl-ethylamino]-methyl}-3-methyl-butyl)-carbamicacid tert-butyl ester

[0089]

[0090] A solution consisting of 1.00 g (4.64 mmol)N-tert-butoxycarbonyl-L-leucinal and 1.52 g (4.64 mmol)2-amino-3-(4-benzyloxy-phenyl)-N-tert-butyl-propionamide (Example 2,Step C) in 20 mL methanol containing 1% acetic acid was stirred at 25°C. for 20 minutes, at which time a solution of 0.614 g (9.29 mmol)sodium cyanotrihydridoborate (Aldrich, Milwaukee, Wis.) in 12 mLmethanol was added and the resulting mixture stirred for 24 hours. Thereaction was quenched by the addition of 3 M HCl in 3 mL incrementsuntil the vigorous bubbling subsided. The mixture was neutralized withsaturated NaHCO₃ solution then extracted with three 50 mL portions ofethyl acetate. The combined extracts were washed with 100 mL brine,dried over anhydrous sodium sulphate, and evaporated at reduced pressureto give a clear, colorless oil which was chromatographed on a silica gelcolumn using 30% to 40% ethyl acetate:hexane to give 735 mg (30%) of[S-(R*,S*)]-(1-{[2-(4-benzyloxy-phenyl)-1-tert-butylcarbamoyl-ethylamino]-methyl}-3-methyl-butyl)-carbamicacid tert-butyl ester as a clear foam. APCI-MS: m/z 526.3 [MH⁺].Microanalysis for C₃₁H₄₇N₃O₄.0.25 H₂O:

[0091] Calculated: C, 70.22; H, 9.03; N, 7.92.

[0092] Found: C, 70.27; H, 9.09; N, 7.78.

[0093] Step E

(S,S)-2-(2-Amino-4-methyl-pentylamino)-3-(4-benzyloxy-phenyl)-N-tert-butyl-propionamide

[0094]

[0095] A solution of 850 mg (1.6 mmol)(1-{[2-(4-benzyloxy-phenyl)-1-tert-butylcarbamoyl-ethylamino]-methyl}-3-methyl-butyl)-carbamicacid tert-butyl ester in 6 mL dichloromethane was treated with 2 mL2,2,2-triflouroacctic acid and the resulting solution stirred 1 hour at25° C. The mixture was concentrated at reduced pressure to a viscousamber oil to which was dissolved in 8 mL diethyl ether, and treated with3 mL of diethyl ether which had been saturated with hydrogen chloridegas. The solvent was removed under reduced pressure, and the pale yellowoil thus obtained((S,S)-2-(2-amino-4-methyl-pentylamino)-3-(4-benzyloxy-phenyl)-N-tert-butyl-propionamide)was used directly in the next reaction.

[0096] Step F

Azepane-1-carbonyl chloride

[0097]

[0098] Azepane-1-carbonyl chloride was made according to the procedurereported in Tetrahedron Lett., 1994;35:839.

[0099] Step G

[0100] A solution of 746 mg (1.6 mmol)2-(2-amino-4-methyl-pentylamino)-3-(4-benzyloxy-phenyl)-N-tert-butyl-propionamide,0.408 mL (3.1 mmol) hexahydro-azepinyl carbamoyl chloride, 20 mg (0.16mmol) 4-dimethylaminopyridine (Aldrich, Milwaukee, Wis.), 0.818 mL (4.7mmol) N,N-diisopropylethylamine (Aldrich, Milwaukee, Wis.), in 10 mL ofbenzene was heated under reflux for 16 hours, cooled to 25° C. andfiltered, and the solids washed with benzene. The filtrate wasconcentrated under reduced pressure, and the residue thus obtained wasdissolved in 100 mL ethyl acetate, and washed with 100 mL each saturatedNaHCO₃ solution and brine, dried over anhydrous magnesium sulfate, thenconcentrated under reduced pressure to give a pale amber oil that waspurified by column chromatography on silica gel with 1:1 ethylacetate:chloroform as eluant. The clear, colorless oil obtained wasdissolved in 10 mL diethyl ether, and treated with diethyl ether thathad been saturated with hydrogen chloride gas. The precipitate thatformed was collected by filtration, and the solids washed with ethylacetate and dried. The title compound was obtained as 440 mg off-whitesolid (77%); mp 51-57° C. APCI-MS: m/z 551.4 [MH⁺].

EXAMPLE 3 [S-(R*,R*)]-2-Dimethylamino-4-methyl-pentanoic acid[2-(4-benzyloxy-phenyl)-1-morpholin-4-ylmethyl-ethyl]-amide

[0101]

[0102] Step A

(S)-2-Dimethylamino-4-methyl-pentanoic acid

[0103]

[0104] (S)-2-Dimethylamino-4-methyl-pentanoic acid was synthesizedaccording to the procedure described in J. Chem. Soc., 1950:1342-1345.

[0105] Step B

N-tert-Butoxycarbonyl-O-benzyl-L-tyrosinal

[0106]

[0107] N-tert-Butoxycarbonyl-O-benzyl-L-tyrosinal was synthesizedaccording to the procedure published in Thompson, et al., TetrahedronLett., 1990;31:6819-6822.

[0108] Step C

[2-(4-Benzyloxy-phenyl)-1-morpholin-4-ylmethyl-ethyl]-carbamic acidtert-butyl ester

[0109]

[0110] A solution consisting of 2.86 g (8 mmol)N-tert-butoxycarbonyl-O-benzyl-L-tyrosinal and 0.701 mL (8 mmol)morpholine (Aldrich, Milwaukee, Wis.) in 36 mL methanol containing 1%acetic acid was stirred at 25° C. for 20 minutes, at which time asolution of 1.06 g (16 mmol) sodium cyanotrihydridoborate (Aldrich,Milwaukee, Wis.) in 12 mL methanol was added and the resulting mixturestirred for 24 hours. The reaction was quenched by the addition of 3 MHCl in 3 mL increments until the vigorous bubbling subsided. The mixturewas neutralized with saturated NaHCO₃ solution then extracted with three100 mL portions of ethyl acetate. The combined extracts were washed with100 mL brine, dried over anhydrous sodium sulphate, and evaporated atreduced pressure to give a clear, colorless oil which waschromatographed on a silica gel column using ethyl acetate:hexane, 1:1as eluant. The A white, waxy solid was recrystallized from2,2,4-trimethylpentane to give 1.53 g (45%) of[2-(4-benzyloxy-phenyl)-1-morpholin-4-ylmethyl-ethyl]-carbamic acidtert-butyl ester as soft white crystals; mp 98-99° C. APCI-MS m/z 427.2[MH⁺]. Microanalysis for C₃₄H₄₇N₃O.0.25H₂O:

[0111] Calculated: C, 78.80; H, 9.24; N, 8.11.

[0112] Found: C, 78.64; H, 9.56; N, 7.95.

[0113] Step D

(S)-2-(4-Benzyloxy-phenyl)-1-morpholin-4-ylmethyl-ethylamine

[0114]

[0115] A solution of 1.41 g (3.3 mmol)[2-(4-benzyloxy-phenyl)-1-morpholin-4-ylmethyl-ethyl]-carbamic acidtert-butyl ester in 5 mL dichloromethane was treated with 1.5 mL2,2,2-triflouroacetic acid and the resulting solution stirred 16 hoursat 25° C. The mixture was concentrated at reduced pressure to a viscousamber oil to which was added 50 mL saturated aqueous sodium bicarbonatesolution and the resulting mixture extracted with three 50 mL portionsof ethyl acetate. The combined extracts were washed with 100 mL brine,dried over anhydrous sodium sulfate and concentrated at reduced pressureaffording a soft solid, that was recrystallized from2,2,4-trimethylpentane containing about 5% ethyl acetate to afford 804mg (77%) of (S)-2-(4-benzyloxy-phenyl)-1-morpholin-4-ylmethyl-ethylamineas light tan crystals; mp 180-182° C. MS: m/z 327.2 [MH⁺].

[0116] Step E

[0117] (S)-2-Dimethylamino-4-methyl-pentanoic acid (55.3 mg, 0.347 mmol)was dissolved in dry DMF (3 mL) under nitrogen atmosphere and cooled to0° C. in an ice-water bath. To this solution were added in successionN,N-diisopropylethylamine (0.180 mL, 1.04 mmol) and solidO-benzotriazol-1-yl-N,N,N′,N′-tetramethyluronium hexafluorophosphate(132 mg, 0.347 mmol, Novabiochem, La Jolla, Calif.). The resultingreaction mixture was stirred at that temperature for 30 minutes,(S)-2-(4-benzyloxy-phenyl)-1-morpholin-4-ylmethyl-ethylamine (153 g,0.347 mmol, Example 3, Step D) was then added. After an additional 60minutes stirring at 0° C., the reaction mixture was mixed with 60 mL ofdiethyl ether, and the resulting mixture was successively washed withsaturated aqueous NaHCO₃ solution and brine and then dried over Na₂SO₄.The solution was concentrated in vacuo, an oil was obtained. The crudeproduct was purified by chromatography (silica gel, 10% methanol inchloroform) to give the title compound as a white solid (90 mg, 55%); mp113-114° C. APCI-MS: m/z 468.4 (MH⁺).

EXAMPLE 4 [S-(R*,R*)]-Azepane-1-carboxylic acid{1-[2-(4-benzyloxy-phenyl)-1-morpholin-4-ylmethyl-ethylcarbamoyl]-3-methyl-butyl}-amide

[0118]

[0119] A solution of 87 mg (0.34 mmol) of(S)-2-[(azepane-1-carbonyl)-amino]-4-methyl-pentanoic acid (Example 1,Step B) and 0.131 mL (1.2 mmol) 4-methylmorpholine was stirred at 25° C.for 30 minutes, after which 136 mg (0.36 mmol)O-benzotriazol-1-yl-N,N,N′,N′-bis(tetramethylene)uroniumhexafluorophosphate in 4 mL dry DMF was added. The resulting solutionwas cooled in an ice-bath and 150 mg (0.34 mmol)2-(4-benzyloxy-phenyl)-1-morpholin-4-ylmethyl-ethylamine (Example 3,Step D) was added. The resulting solution was warmed to 25° C. andstirred for 30 minutes. The mixture was then poured into 35 mL diethylether, and washed sequentially with 25 mL each of 2.5% aqueous HClsolution, brine, saturated aqueous sodium bicarbonate solution, andfinally twice with brine. The mixture was then dried over anhydroussodium sulfate and concentrated at reduced pressure to give an amber oilthat was purified by preparative thin-layer chromatography on a 1000 μmsilica gel plate with 50:50:1 ethyl acetate:hexane:methanol as eluant togive 46 mg (24%) of the title compound as a pale amber glass. APCI-MS:m/z 565.5 [MH⁺]. Microanalysis for C₃₃H₄₈N₄O₄.0.25EtOAc:

[0120] Calculated: C, 69.59; H, 8.59; N, 9.55.

[0121] Found: C, 69.70; H, 8.67; N, 9.39.

EXAMPLE 5 [S-(R*,R*)]Azepane-1-carboxylic acid{1-[2-(4-benzyloxy-phenyl)-1-(tert-butylamino-methyl)-ethylcarbamoly]-3-methyl-butyl}-amidetrifloroacetate salt (1:1)

[0122]

[0123] Step A

(S)-[2-(4-Benzyloxy-phenyl)-1-(tert-butylamino-methyl)-ethyl]-carbamicacid tert-butyl ester

[0124]

[0125] A solution consisting of 2.86 g (8 mmol)N-tert-butoxycarbonyl-O-benzyl-L-tyrosinal (Example 4, Step A) and 0.846mL (8 mmol) tert-butyl amine (Aldrich, Milwaukee, Wis.) in 36 mLmethanol containing 1% acetic acid was stirred at 25° C. for 20 minutes,at which time a solution of 1.06 g (16 mmol) sodiumcyanotrihydridoborate (Aldrich, Milwaukee, Wis.) in 12 mL methanol wasadded and the resulting mixture stirred for 48 hours. The reaction wasquenched by the addition of 3 M HCl in 3 mL increments until thevigorous bubbling subsided. The mixture was neutralized with saturatedNaHCO₃ solution then extracted with three 100 mL portions of ethylacetate. The combined extracts were washed with 100 mL brine, dried overanhydrous sodium sulphate and evaporated at reduced pressure to give apale amber oil which was chromatographed on a silica gel column using 1%to 2% methanol in ethyl acetate as eluant to give 604 mg (18%) of thetitle compound as waxy white crystals; mp 77-79° C. APCI-MS m/z 413.2[MH⁺].

[0126] Step B

(S)-3-(4-Benzyloxy-phenyl)-N¹-tert-butyl-propane-1,2-diamine

[0127]

[0128] A solution of 1.41 g (3.3 mmol)[2-(4-benzyloxy-phenyl)-1-morpholin-4-ylmethyl-ethyl]-carbamic acidtert-butyl ester in 5 mL dichloromethane was treated with 1.5 mL2,2,2-triflouroacetic acid and the resulting solution stirred 16 hoursat 25° C. The mixture was concentrated at reduced pressure to a viscousamber oil to which was added 50 mL saturated aqueous sodium bicarbonatesolution and the resulting mixture extracted with three 50 mL portionsof ethyl acetate. The combined extracts were washed with 100 mL brine,dried over anhydrous sodium sulfate and concentrated at reduced pressureaffording a soft solid, that was recrystallized from2,2,4-trimethylpentane containing about 5% ethyl acetate to afford 804mg (77%) of the title compound as light tan crystals; mp 180-182° C.APCI-MS m/z 382.2 [MH⁺].

[0129] Step C

[0130] A solution of 200 mg (0.79 mmol) of(S)-2-[(azepane-1-carbonyl)-amino]-4-methyl-pentanoic acid (Example 1,Step B) and 0.260 mL (2.4 mmol) 4-methylmorpholine in 4 mL DMF wasstirred at 25° C. for 0.5 hour, and 298 mg (0.79 mmol)O-benzotriazol-1-yl-N,N,N′,N′-bis(tetramethylene)uroniumhexafluorophosphate was added, and the resulting solution cooled in anice-bath and stirred 30 minutes, and 236 mg (0.79 mmol) of[2-(4-benzyloxy-phenyl)-1-(tert-butylamino-methyl)-ethylamine was addedand the resulting solution warmed to 25° C. and stirred 30 minutes. Themixture was poured into 50 mL diethyl ether, and washed with 55 mLsaturated aqueous sodium bicarbonate solution, and finally twice with 75mL brine, and dried over anhydrous sodium sulfate and concentrated atreduced pressure to give a pale amber oil that was purified by columnchromatography on silica gel with 3% methanol in chloroform as eluant togive 117 mg (27%) of the title compound as a white foam. APCI-MS m/z565.5 [MH⁺]. Microanalysis for C₃₃H₅₀N₄O₃.TFA.0.5 H₂O:

[0131] Calculated: C, 62.39; H, 7.78; N, 8.31; H₂O, 1.34.

[0132] Found: C, 62.45; H, 8.17; N, 8.58; H₂O, 1.57.

EXAMPLE 6 (S,S)-Azepane-1-carboxylic acid{1-[2-(4-benzyloxy-phenyl)-1-diethylaminomethyl-ethylcarbamoyl]-3-methyl-butyl}-amide

[0133]

[0134] Step A

(S)-[1(4-Benzyloxy-benzyl)-2-diethylamino-ethyl]-carbamic acidtert-butyl ester

[0135]

[0136] A solution consisting of 2.86 g (8 mmol)N-tert-butoxycarbonyl-O-benzyl-L-tyrosinal (Example 3, Step B) and 0.832mL (8 mmol) diethyl amine in 36 mL methanol containing 1% acetic acidwas stirred at 25° C. for 20 minutes, at which time a solution of 1.06 g(16 mmol) sodium cyanotrihydridoborate (Aldrich, Milwaukee, Wis.) in 12mL methanol was added and the resulting mixture stirred for 48 hours.The reaction was quenched by the addition of 3 M HCI in 3 mL incrementsuntil the vigorous bubbling subsided. The mixture was neutralized withsaturated NaHCO₃ solution then extracted with three 100 mL portions ofethyl acetate. The combined extracts were washed with 100 mL brine,dried over anhydrous sodium sulphate and evaporated at reduced pressureto give an amber oil which was chromatographed on a silica gel columnusing 50:50:1 ethyl acetate:hexane:methanol as eluant to give 236 mg(7%) of (S)-[1-(4-benzyloxy-benzyl)-2-diethylamino-ethyl]-carbamic acidtert-butyl ester as a clear, colorless oil. APCI-MS: m/z 413.2 [MH⁺].

[0137]¹H NMR (CDCl₃): 7.40-7.28 (5H, m), 7.08 (2H, d, J=8.5 Hz), 6.86(2H, d, J=8.5 Hz), 5.00 (2H, s), 3.77 (1H, bs), 2.77-2.37 (7H, m), 1.38(9H, s), 0.96 (6H, bs).

[0138] Step B

(S)-3-(4-Benzyloxy-phenyl)-N¹,N¹-diethyl-propane-1,2-diamine

[0139]

[0140] A solution of 171 mg (0.4 mmol)[2-(4-benzyloxy-phenyl)-1-(diethylamino-methyl)-ethyl]-carbamic acidtert-butyl ester in 2 mL dichloromethane was treated with 0.5 mL2,2,2-triflouroacetic acid and the resulting solution stirred 3 hours at25° C. The mixture was concentrated at reduced pressure to a viscousamber oil to which was added 30 mL saturated aqueous sodium bicarbonatesolution and the resulting mixture extracted with three 20 mL portionsof ethyl acetate. The combined extracts were washed with two 50 mLportions of brine, dried over anhydrous sodium sulfate, and concentratedat reduced pressure and the residue purified by column chromatographyusing ethyl acetate containing 1% methanol as eluent. There was obtained112 mg (90%) of(S)-3-(4-benzyloxy-phenyl)-N¹,N¹-diethyl-propane-1,2-diamine as a clear,colorless oil. APCI-MS: m/z 313.2 [MH⁺].

[0141] 1H NMR (CDCl₃): 7.37-7.25 (5H, m), 7.07 (2H, d, J=8.06 Hz), 6.85(2H, d, J=7.8 Hz), 4.98 (2H, s), 3.05 (1H, bs), 2.67-2.24 (6H, m), 0.96(6H, t).

[0142] Step C

[0143] A solution of 53 mg (0.20 mmol) of(S)-2-[(azepane-1-carbonyl)-amino]-4-methyl-pentanoic acid (Example 1,Step B) and 0.079 mL (7.2 mmol) 4-methylmorpholine in 4 mL DMF wasstirred at 25° C. for 0.5 hour, and 82 mg (0.21 mmol)0-benzotriazol-1-yl-N,N,N′,N′-bis(tetramethylene)uroniumhexafluorophosphate was added. The resulting solution was cooled in anice-bath and stirred 30 minutes, after which 64 mg (0.20 mmol) of(S)-3-(4-benzyloxy-phenyl)-N¹,N¹-diethyl-propane-1,2-diamine in 2 mL DMFwas added. The resulting solution was warmed to 25° C. and stirred for30 minutes. The mixture was then poured into 30 mL diethyl ether, andwashed with 25 mL each of 5% HCl solution, brine, saturated aqueoussodium bicarbonate solution, and finally twice with brine. The mixturewas dried over anhydrous sodium sulfate and concentrated at reducedpressure to give a clear, colorless oil that was purified by preparativethin-layer chromatography on a 500 gm silica gel plate using 50:50:1ethyl acetate:hexane:methanol as eluant to give 21.2 mg (19%) of thetitle compound as a clear, colorless oil. APCI-MS: m/z 551.5 [MH⁺].Microanalysis for C₃₃H₄₈N₄O₃.0.5 H₂O:

[0144] Calculated: C, 70.81; H, 9.18; N, 10.01; H₂O, 1.61.

[0145] Found: C, 70.64; H, 8.97; N, 9.73; H₂O, 1.29.

EXAMPLE 7(S)-2-(2-Amino-4-methyl-pentylamino)-3-(4-benzyloxy-phenyl)-N-tert-butyl-propionamidedihydrochloride

[0146]

[0147] Step A

(S)-2-Amino-3-(4-benzyloxy-phenyl)-N-tert-butyl-propionamidemonohydrochloride

[0148]

[0149] To a solution of(S)-2-amino-3-(4-benzyloxy-phenyl)-N-tert-butyl-propionamide (Example 2,Step C) in ether was added excess amount of ethereal HCl, solid formedwas isolated via filtration, subsequent drying under vacuum gave titlecompound as a yellow solid.

[0150] Step B

1-{[2-(4-Benzyloxy-phenyl)-1-tert-butylcarbamoyl-ethylamino]-methyl}-3-methyl-butyl)-carbamic acid tert-butyl ester

[0151] (S)-2-Amino-3 -(4-benzyloxy-phenyl)-N-tert-butyl-propionamidemonohydrochloride (1.68 g, 4.65 mmol, Example 7, Step A) and BOC-Leu-CHO((tert-butoxy)-N-[1-(2-methylpropyl)-2-oxoethyl]carboxamide) (1.00 g,4.65 mmol, Peninsula Laboratories, Belmont, Calif.) were mixed in CH₂Cl₂(25 mL). After stirring at ambient temperature under nitrogen atmospherefor 30 minutes, the solution was cooled to 0° C. in an ice-water bath.To this solution was added sodium triacetoxyborohydride (1.50 g, 6.98mmol). The resulting reaction mixture was stirred for, in succession, 30minutes at 0° C. and 12 hours at ambient temperature. Twenty-fivemilliliters of saturated aqueous NaHCO₃ solution was added to thereaction mixture, and the resulting mixture was stirred for 5 minutes.The two layers were separated, and the aqueous layer was extracted withCH₂Cl₂ (2×30 mL). The combined organic solution was dried over Na₂SO₄.The solution was concentrated in vacuo affording a viscous oil. Thereaction gave two diastereomers which were purified twice by columnchromatography (eluant used in first column chromatography: 30% EtOAc-4%MeOH-66% hexanes; eluant used in second column chromatography: 100%acetonitrile). When the solvent system, 30% EtOAc-66% benzene-4% MeOH,was used as eluant. The two diastereomers gave different R_(f) values onTLC (0.68 and 0.53). The compound with a R_(f) value of 0.68 wasisolated by preparative plate as a yellow oil (0.3 g) and used in usedstep.

[0152] Step C

[0153] To a solution of(1-{[2-(4-benzyloxy-phenyl)-1-tert-butylcarbamoyl-ethylamino]-methyl}-3-methyl-butyl)-carbamicacid tert-butyl ester (0.4 g, 0.76 mmol, Example 7, Step B) in CH₂Cl₂(7.5 mL) was added trifluoroacetic acid (2.5 mL) at ambient temperatureunder nitrogen atmosphere. The resulting reaction mixture was stirredfor 25 minutes, then concentrated in vacuo. The viscous oil obtained wasdissolved in 40 mL of CH₂Cl₂ and successively washed with saturatedaqueous NaHCO₃ solution (2×40 mL), brine (2×40 mL), and was dried overNa₂SO₄. The CH₂Cl₂ solution of free amine was concentrated in vacuo toafford the crude product as a viscous oil which was then dissolved in 10mL of ethyl ether. To this solution was added saturated HCl solution inethyl ether until no more white precipitate formed. Filtration anddrying overnight under vacuum afforded 0.23 g (61 %) of titled compoundas a yellow solid; mp 228-230° C. APCI-MS m/z 426.1 (MH⁺).

EXAMPLE 82-(2-Amino-4-methyl-pentylamino)-3-(4-benzyloxy-phenyl)-N-tert-butyl-propionamidedihydrochloride

[0154]

[0155] Step A

(1-{[2-(4-Benzyloxy-phenyl)-1-tert-butylcarbamoyl-ethylamino]-methyl}-3-methyl-butyl)-carbamicacid tert-butyl ester

[0156]

[0157] (S)-2-Amino-3-(4-benzyloxy-phenyl)-N-tert-butyl-propionamidemonohydrochloride (1.68 g, 4.65 mmol, Example 7, Step A) and BOC-Leu-CHO((tert-butoxy)-N-[1-(2-methylpropyl)-2-oxoethyl]carboxamide) (1.00 g,4.65 mmol, Peninsula Laboratories, Belmont, Calif.) were mixed in CH₂Cl₂(25 mL). After stirring at ambient temperature under nitrogen atmospherefor 30 minutes, the solution was cooled to 0° C. in an ice-water bath.To this solution was added sodium triacetoxyborohydride (1.50 g, 6.98mmol). The resulting reaction mixture was stirred for, in succession, 30minutes at 0° C. and 12 hours at ambient temperature. Twenty-fivemilliliters of saturated aqueous NaHCO₃ solution was added to thereaction mixture, and the resulting mixture was stirred for 5 minutes.The 2 layers were separated, and the aqueous layer was extracted withCH₂Cl₂ (2×30 mL). The combined organic solution was dried over Na₂SO₄.The solution was concentrated in vacuo affording a viscous oil. Thereaction gave two diastereomers which were purified twice by columnchromatography (eluant used in first column chromatography: 30% EtOAc-4%MeOH-66% hexanes; eluant used in second column chromatography: 100%acetonitrile). When the solvent system, 30% EtOAC-66% benzene-4% MeOH,was used as eluant, The two diastereomers gave different R_(f) values onTLC (0.68 and 0.53). The compound with a R_(f) value of 0.53 wasisolated by preparative plate as a yellow oil (0.27 g) and used in usedstep.

[0158] Step B

[0159] To a solution of(1-{[2-(4-benzyloxy-phenyl)-1-tert-butylcarbamoyl-ethylamino]-methyl}-3-methyl-butyl)-carbamicacid tert-butyl ester (0.4 g, 0.76 mmol, Example 8, Step A) in CH₂Cl₂(7.5 mL) was added trifluoroacetic acid (2.5 mL) at ambient temperatureunder nitrogen atmosphere. The resulting reaction mixture was stirredfor 25 minutes, then concentrated in vacuo. The viscous oil obtained wasdissolved in 40 mL of CH₂Cl₂ and successively washed with saturatedaqueous NaHCO₃ solution (2×40 mL), brine (2×40 mL), and was dried overNa₂SO₄. The CH₂Cl₂ solution of free amine was concentrated in vacuo toafford the crude product as a viscous oil which was then dissolved in 10mL of ethyl ether. To this solution was added saturated HCl solution inethyl ether until no more white precipitate formed. Filtration anddrying overnight under vacuum afforded 0.15 g (59%) of titled compoundas a yellow solid; mp 228-230° C. APCI-MS m/z 426.1 (MH⁺).

EXAMPLE 9 Biological Assays

[0160] Measurement of N-type Ca²⁺Channel Blocking Potencies of Compoundsin IMR32 Cells Using the Fluorescent Ca²⁺Indicator Indo-1

[0161] IMR-32 cells are a human tumoral cell line of neural origin. TheIMR-32 cell line has been shown to contain both N- and L-type voltagesensitive calcium channels. Calcium flux into these cells may be inducedby stimulation with elevated potassium concentrations. The L-channelcomponent of calcium flux may be blocked by adding 5 μM nitrendipine.The remaining component of calcium entry into the IMR-32 cells is due tocalcium flux through N-type calcium channels. Intracellular calciumconcentrations are measured using the fluorescent calcium indicatorIndo-1. The effect of drug concentration on calcium uptake is studied.

[0162] The IMR-32 cell line was obtained from the American Type CultureCollection (Rockville, Md.). Cells were grown in Eagle's MinimumEssential Medium with Earle's salts supplemented with 10% fetal bovineserum, 2 mM L-Gln and antibiotic/antimicotic mixture (Gibco). Atapproximately 80% confluency, differentiation was induced by theaddition of 1 mM dibutyryl cAMP and 2.5 μM bromodeoxyuridine to themedium. After 7 to 13 days of differentiation, cells were detached using0.5 mM EDTA and loaded with 5 μM Indo-1 acetoxymethyl ester (MolecularProbes, Eugene, Oreg.) at 300° C. for 45 minutes. Loaded cells werewashed twice, resuspended (−10⁷ cells/mL) in assay buffer (10 mMHEPES/Tris pH 7.4 in Hank's Balanced Salt Solution without bicarbonateor phenol red containing 0.5% bovine serum albumin) and kept on iceuntil use. Fluorescence measurements were carried out in a PhotonTechnology International (PTI, South Brunswick, N.J.) Model RF-F3004spectrofluorometer with dual emission monochromators using excitation at350 nm and emission at 400 and 490 nm. The instrument was equipped witha thermostated cuvette holder with stirring capabilities as well as witha computer-controlled pump that allowed for reagent addition duringmeasurement. Instrument control and data collection was done by PTI'sOSCAR software running on an IBM compatible computer. Differentconcentrations of the test compounds (60 μL in dimethyl sulfoxide) wereadded to 5.94 mL of assay buffer containing approximately 3×10⁶ loadedcells, and 5 μM Nitrendipine (in 30 μL EtOH) to block L-typeCa²⁺channels. Samples were incubated for 10 minutes at 30° C. and thenaliquoted into three 10×10 mm disposable acrylic cuvettes. Emissionsignals at 400 and 409 nm were acquired from each cuvette at 30° C. for50 seconds. At 20 seconds after the start of reading, cells weredepolarized by the addition of 160 μL of stimulation solution (1M KCl,68 mM CaCl₂) to the cuvette via the computer-controlled pump. Ratio ofdual emission signals (400 nm/409 nm), which is proportional tointracellular Ca²⁺concentration, was plotted against time, and thedifference between maximal response after stimulation and basal value(before stimulation) was determined. Values obtained in this way wereplotted as a function of drug concentration. IC₅₀ values of testcompounds were calculated by fitting a 4-parameter logistic function tothe data using the least squares method.

[0163] The following as the results for the IMR32 and FLP150 assays:TABLE 1 Compound of IMR32 Example No. IC₅₀ (μM) 1 0.21 2 0.19 3 48% @ 10μM 6% @ 1 μM 4 1.2  5 1.1  6 1.1  7 1.2  8 1  

[0164] Audiogenic Seizure Model in DBA/2 Mice:

[0165] Compounds of the present invention were dissolved in water using10% (weight/volume)Emulphor (GAF Corp., Wayne, N.J.) surfactant.Substances were admimistered by intravenous injection into theretro-orbital venous sinus. All testing was performed 15 minutes or 45minutes after drug injection. All the male mice, 3 to 4 weeks old, wereobtained from Jackson Laboratories, Bar Harbour, Me. Immediately beforeanticonvulsant testing, mice were placed upon a wire mesh, 4 inchessquare suspended from a steel rod. The square was slowly invertedthrough 180 degrees, and the mice were observed for 30 seconds. Anymouse falling from the wire mesh was scored as ataxic.

[0166] Mice were placed into an enclosed acrylic plastic chamber (21 cmheight, approximately 30 cm diameter) with a high-frequency speaker (4cm diameter) in the center of the top lid. An audio signal generator(Protek model B-810) was used to produce a continuous sinusoidal tonethat was swept linearly in frequency between 8 kHz and 16 kHz once each10 msec. The average sound pressure level (SPL) during stimulation wasapproximately 100 dB at the floor of the chamber. Mice were placedwithin the chamber and allowed to acclimatize for 1 minute. DBA/2 micein the vehicle-treated group responded to the sound stimulus (applieduntil tonic extension occurred, or for a maximum of 60 seconds) with acharacteristic seizure sequence consisting of wild running followed byclonic seizures, and later by tonic extension, and finally byrespiratory arrest and death in 80% or more of the mice. Invehicle-treated mice, the entire sequence of seizures to respiratoryarrest lasts approximately 15 to 20 seconds.

[0167] The incidence of all the seizure phases in the drug-treated andvehicle-treated mice was recorded, and the occurrence of tonic seizureswere used for calculating anticonvulsant ED values by probit analysis.Mice were used only once for testing at each time and dose point.

[0168] Compound 2 was found to give 100% protection at 30 mg/kg at 15minutes posttreatment time, and Compound 1 gave 80% protection at 30mg/kg at 15 minutes posttreatment time. TABLE 2 Compound of Time PostNumber of Mice Example No. Dose Treatment Protected from Tonic Number(mg/kg, IV) (minutes) Convulsions* 1 30 15 4/5 1 30 45 2/5 1 10 15 1/5 230 15 5/5 2 30 45 1/5 2 10 15 1/5 7 10 15 2/5 7 10 45 1/5 8 10 15 0/5 810 45 0/5

What is claimed is:
 1. A compound having structural Formula I:

wherein R¹ is H or methyl, R² is H, azepanylcarbonyl, C₁-C₇ alkyl,—(CH₂)_(n)-phenyl, wherein the phenyl is unsubstituted or substitutedwith C₁-C₄-alkyl, C₁-C₄ alkoxy, or halo, R³ is C₁-C₅ alkyl, X is —NR⁴R⁵or —OR⁷, R⁴ and R⁵ are independently H, C₁-C₅ alkyl, or R⁴ and R⁵together with the nitrogen to which they are both bound form:

 R⁶ is —(CH₂)_(n)-phenyl, wherein the phenyl is unsubstituted orsubstituted with C₁-C₄ alkyl, C₁-C₄ alkoxy, or halo, A and B areindependently —CO— or —CH₂—, provided that A and B are not both —CO—, R⁷is C₁-C₅ alkyl, Z is —CH₂—, —O—, —S—, or —N(R⁸)—, R⁸ is H or C₁-C₆alkyl, and n is 1 or
 2. 2. The compound according to claim 1, wherein R⁴and R⁵ are independently H, C₁-C₅-alkyl, or R⁴ and R⁵ taken togetherwith the nitrogen to which they are both bound form 1-pyrrolidinyl,1-piperidinyl, or 1-azepanyl, or R⁴ and R⁵ taken together with thenitrogen to which they are both bound are:

and Z is —S— or —O—.
 3. A compound according to claim 1 selected fromthe group consisting of2-((2-(Azaperhydroepinylcarbonylamino)-4-methylpentyl)amino)-N-(tert-butyl)-3-(4-(phenylmethoxy)phenyl)-(2S)-propanamide;[S-(R*,R*)]-Azepane-1-carboxylic acid{1-[2-(4-benzyloxy-phenyl)-1-tert-butoxy-methyl-ethylcarbamoyl]-3-methyl-butyl}-amide;[S-(R*,R*)]Azepane-1-carboxylic acid(1-{[2-(4-benzyloxy-phenyl)-1-tert-butyl-carbamoyl-ethylamino]-methyl}-3-methyl-butyl)-amide;[S-(R*,R*)]-2-Dimethylamino-4-methyl-pentanoic acid[2-(4-benzyloxy-phenyl) 1-morpholin-4-ylmethyl-ethyl]-amide;[S-(R*,R*)]-Azepane-1-carboxylic acid{1-[2-(4-benzyloxy-phenyl)-1-morpholin-4-ylmethyl-ethylcarbamoyl]-3-methyl-butyl}-amide;[S-(R*,R*)]Azepane-1-carboxylic acid{1-[2-(4-benzyloxy-phenyl)-1-(tert-butyl-amino-methyl)-ethylcarbamoyl]-3-methyl-butyl}-amide;(S,S)-Azepane-1-carboxylic acid{1-[2-(4-benzyloxy-phenyl)-1-diethylaminomethyl-ethylcarbamoyl]-3-methyl-butyl}-amide;(S)-2-(2-Amino-4-methyl-pentylamino)-3-(4-benzyloxy-phenyl)-N-tert-butyl-propionamide;and2-(2-Amino-4-methyl-pentylamino)-3-(4-benzyloxy-phenyl)-N-tert-butyl-propionamide.4. A therapeutic composition comprising an effective calcium channelblocking amount of the compound of claim 1 with a pharmaceuticallyacceptable carrier.
 5. A therapeutic composition comprising an effectiveamount of the compound of claim 0 with a pharmaceutically acceptablecarrier.
 6. A method of blocking calcium channels, the method comprisingadministering to a mammal in need of calcium channel blocking, atherapeutically effective amount of a compound of claim 1 to blockcalcium channels.
 7. A method of blocking calcium channels, the methodcomprising administering to a mammal in need of calcium channelblocking, a therapeutically effective amount of a compound of claim 3 toblock calcium channels.
 8. A method of treating stroke, the methodcomprising administering to a mammal having a stroke a therapeuticallyeffective amount of a compound of claim
 1. 9. A method of preventing astroke, the method comprising administering to a mammal at risk ofhaving a stroke a therapeutically effective amount of a compound ofclaim
 1. 10. A method of treating stroke, the method comprisingadministering to a mammal having a stroke a therapeutically effectiveamount of a compound of claim
 3. 11. A method of preventing a stroke,the method comprising administering to a mammal at risk of having astroke a therapeutically effective amount of a compound of claim
 3. 12.A method of treating cerebral ischemia, the method comprisingadministering to a mammal having cerebral ischemia a therapeuticallyeffective amount of a compound of claim
 1. 13. A method of treatingcerebral ischemia, the method comprising administering to a mammalhaving cerebral ischemia a therapeutically effective amount of acompound of claim
 3. 14. A method of treating head trauma, the methodcomprising administering to a mammal having head trauma atherapeutically effective amount of a compound of claim
 1. 15. A methodof treating head trauma, the method comprising administering to a mammalhaving head trauma a therapeutically effective amount of a compound ofclaim
 3. 16. A method of treating epilepsy, the method comprisingadministering to a mammal having epilepsy a therapeutically effectiveamount of a compound of claim
 1. 17. A method of treating epilepsy, themethod comprising administering to a mammal having epilepsy atherapeutically effective amount of a compound of claim
 3. 18. A methodof treating pain, the method comprising administering to a mammal havingpain a therapeutically effective amount of a compound of claim
 1. 19. Amethod of treating pain, the method comprising administering to a mammalhaving pain a therapeutically effective amount of a compound of claim 3.20. A method of inhibiting N-type calcium channels in a cell, comprisingcontacting a cell having one or more N-type calcium channels with acompound of claim
 1. 21. A method of inhibiting N-type calcium channelsin a cell, comprising contacting a cell having one or more N-typecalcium channels with a compound of claim 3.